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Journal of Bacteriology, October 2005, p. 6779-6788, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6779-6788.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus: Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

Junichi Miyazaki,¹ Kuniko Asada,¹ Shinya Fushinobu,² Tomohisa Kuzuyama,¹ and Makoto Nishiyama^1*

Biotechnology Research Center,¹ Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan²

Received 28 May 2005/ Accepted 18 July 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related ß-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Homoisocitrate dehydrogenase (HICDH) is the third enzyme involved in lysine biosynthesis through -aminoadipate (19) and is a member of a family of ß-decarboxylating dehydrogenases that includes isocitrate dehydrogenase (ICDH) in the tricarboxylic acid cycle, 3-isopropylmalate dehydrogenase (IPMDH) in leucine biosynthesis, and tartrate dehydrogenase in vitamin production (2). It has been suggested that these enzymes diverged from a common ancestral ß-decarboxylating dehydrogenase (4, 9, 34).

We have shown that in an extremely thermophilic bacterium, Thermus thermophilus HB27, lysine is synthesized through -aminoadipate, although lysine is synthesized through diaminopimelate in most bacteria (13, 18, 20, 22, 23, 31). We have previously characterized HICDH from T. thermophilus HB27 (TtHICDH) in detail and have shown that the enzyme has four unique features (19). The first is its substrate specificity. Although TtHICDH is essential for lysine biosynthesis in the bacterium, TtHICDH can catalyze the reaction with isocitrate as a substrate; isocitrate is the substrate of ICDH at a 20-fold-higher efficiency than toward the native substrate, homoisocitrate. This feature contrasts with HICDH from Saccharomyces cerevisiae, which utilizes only homoisocitrate as a substrate (19). Moreover, the substrate specificity is easily altered by a single mutation, Arg85Val, to lose activity for isocitrate but to acquire activity for 3-isopropylmalate, a substrate of IPMDH. The second feature is the unique subunit organization. TtHICDH is a homotetramer, while most ß-decarboxylating dehydrogenases are homodimers. Although a few members of ß-decarboxylating dehydrogenases are reported to be tetramers (26, 27, 33), the interactions necessary for tetramer formation have not been elucidated at all. The third characteristic is the thermotolerance of TtHICDH, which is substantially higher than for IPMDH and ICDH from T. thermophilus. The fourth is the phylogenetic location of this enzyme. TtHICDH is positioned in the deepest branch in the phylogenetic tree constructed for ß-decarboxylating dehydrogenases, suggesting that the enzyme may possess features of the ancestral enzyme. Thus, the crystal structure of TtHICDH will provide a better understanding of a wide variety of biochemical and evolutionary characteristics.

In this paper, we report the crystal structure of TtHICDH. The structural analysis reveals that TtHICDH is stabilized by a dimer-dimer interaction, which was confirmed by a single mutation weakening the tetramerization. Based on its crystal structure and mutational analysis, we also describe possible local conformational changes upon substrate binding in TtHICDH.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Crystallization of TtHICDH. Overexpression and purification of TtHICDH were performed as described previously (19). Crystallization was carried out by the hanging-drop vapor diffusion method (28). Three microliters of 10-mg/ml purified TtHICDH was added to an equal volume of the reservoir solution containing 24% polyethylene glycol 400 (PEG400) and 0.1 M citrate, pH 4.8. Then, 3 µl of the solution containing 100 mM MgCl₂, 5 mM isocitrate or homoisocitrate, and 1 µl of 50 mM CdCl₂ was added to the drop. The mixture was equilibrated against 500 µl of the reservoir solution at 20°C. Crystals were grown in 5 days, with a typical dimension of 0.5 mm by 0.5 mm by 0.05 mm. Before data collection, to avoid freezing, the crystals were transferred to a cryoprotectant solution containing 25% PEG400, 0.1 M MgCl₂, 5 mM CdCl₂, 0.1 M citrate, and 5 mM isocitrate or homoisocitrate.

Data collection. Data were collected with an ADSC-Q210 charge-coupled-device camera on the NW12 station of the Photon Factory-AR, High Energy Accelerator Research Organization (KEK), Japan. All data were collected at 100 K on a single crystal mounted in a cryoloop. Diffraction images were indexed, integrated, and scaled using the HKL2000 program (24). The crystal of TtHICDH belongs to the space group C222₁, with the following unit cell dimensions: a of 60.96 Å, b of 143.24 Å, and c of 177.85 Å. The data collection and processing statistics are given in Table 1. The data collected at the wavelength of 1.85 Å were used for subsequent molecular replacement and crystallographic refinement of TtHICDH.

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TABLE 1. Data collection and refinement statistics

Structure determination and refinement. Molecular replacement was performed with MOLREP (29) in the CCP4 program suite, using the three-dimensional structure of the Ala172Val mutant of IPMDH from T. thermophilus (Protein Data Bank [PDB] identifier [ID], 1G2U) (25) as a model. Model correction in the electron density map was performed with the XtalView program suite (16). Refinement was carried out with CNS1.1 (1). The refinement statistics are given in Table 1. Although electron density for chain A was clearly seen throughout the polypeptide, the density for chain B was partly ambiguous, especially for the two regions of residues 72 to 76 and 264 to 265. The final model of the TtHICDH crystal structure contains all the residues of chain A and most residues of chain B except for residues 72 to 76 and 264 to 265 in an asymmetric unit.

Construction of the mutants. Val135Met was a mutation obtained during the course of a directed evolution screening, which we will publish in the near future elsewhere. To construct a mutant of TtHICDH containing a single mutation, the Val135Met protein, an hicdh gene resulting in the mutation Val135Met was digested with SmaI and EcoRI and the yielded fragment was introduced into the corresponding portion of the TtHICDH expression plasmid pETtHICDH101 (19). The resultant expression plasmid was named pETtHV135M.

The Tyr125Ala mutant was constructed as follows. Two independent PCRs using 19HICN (5'-CCCCAAGCTTCCGGAGGTGAACATATGGCGTACCGGATCTGCTTGATT-3') and Y125A-1 (5'-GCGCCTTTCCTGCTCCACCGCAAGCCCTTC-3') and Y125A-2 (5'-GAAGGGCTTGCGGTGGAGCAGGAAAGGCGC-3') and HICDHCt (5'-TTTGAATTCTTACAGGCTCTTGAGCGCCTC-3') as primer pairs were performed. PCR conditions used were as follows: (step 1) 94°C for 2 min, (step 2) 94°C for 1 min, (step 3) 65°C for 1 min, (step 4) 72°C for 1 min, and (step 5) 72°C for 7 min; steps 2 to 4 were repeated 30 times. A portion of each reaction mixture was mixed and subjected to additional PCR using 19HICN and HICDHCt as primers. The PCR conditions were as follows: (step 1) 94°C for 10 min, (step 2) 94°C for 1 min, (step 3) 65°C for 1 min, (step 4) 72°C for 2 min, and (step 5) 72°C for 7 min; steps 2 to 4 were repeated 30 times. The amplified fragment was digested with HindIII and EcoRI and cloned into pUC119. After verification of the sequence, the NdeI- plus EcoRI-digested fragment of the plasmid was ligated into pET26b (+) (Novagen, Darmstadt, Germany). The resulting plasmid, pETtHY125A, was used for the expression of the Tyr125Ala mutant.

Expression, purification, and determination of quaternary structures of TtHICDH and the mutant. Overexpression and purification of the mutant were carried out by the method for wild-type TtHICDH as described previously (19) except for the heat treatment step, which was carried out at 70°C in place of 85°C for wild-type TtHICDH. Sedimentation equilibrium analysis was carried out using 100 µl of 1-mg/ml enzymes with a Beckman Optima XL-A analytical ultracentrifuge fitted with a Beckman An-60Ti analytical rotor. The subunit organization of each enzyme was analyzed according to the procedure of Van Holde and Baldwin (30) using Origin 2.8 scientific graphing and data analysis and XL-A 2.01 data analysis on a Windows operating system. Partial specific volumes for the wild-type, Val135Met, and Tyr125Ala proteins were calculated to be 0.745, 0.744, and 0.745, respectively, from the amino acid compositions. Molecular mass calibration by gel filtration was carried out using a Superdex200 prepgrade column (Amersham Bioscience, Tokyo, Japan) at a 2.5-ml/min flow rate.

Thermal inactivation of enzymes. The thermal-inactivation temperatures of the enzymes were analyzed as follows. The purified enzymes (0.5 mg/ml) dissolved in buffer (20 mM potassium phosphate, pH 7.5, 0.5 mM EDTA, 0.2 M NaCl) were incubated at various temperatures for 20 min and immediately chilled in ice water. Then, aggregated proteins were removed by centrifugation (23,500 x g for 5 min), and the remaining activity in the supernatant was measured. One microliter of the enzyme solution was added to the reaction mixture (50 mM HEPES, pH 8.0, 200 mM KCl, 5 mM MgCl₂, 1 mM NAD⁺, and 400 µM isocitrate) that was preincubated at 60°C for 5 min. The reaction was monitored at 60°C by following the increase in absorbance at 340 nm.

Kinetic analysis of enzymes. In the kinetic analysis, we used isocitrate instead of homoisocitrate as the substrate for TtHICDH because homoisocitrate is not commercially available at present. Five microliters of enzymes (0.1 mg/ml) was added to 1 ml of assay buffer (50 mM HEPES, pH 7.5, 200 mM KCl, 5 mM MgCl₂, 1 mM NAD⁺, and 0.1 to 4 mM isocitrate), which had been preincubated at 60°C for 5 min. The reaction was monitored at 60°C by following the increase in absorbance at 340 nm. Data were analyzed by Cleland's initial velocity program Hyper (3).

Graphics manipulation. All of Fig. 1, 2, and 3 were generated using the programs MOLSCIPT (14), RASTER3D (17), and XtalView (16).

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FIG. 1. Crystal structure of TtHICDH. A, TtHICDH subunits A and B colored by hot pink and pale pink, respectively, in an asymmetric unit. B, Superposition of two subunits colored by hot pink and pale pink in an asymmetric unit. C, Superposition of TtHICDH (hot and pale pink) on an open-form IPMDH dimer (TtIPMDH, gray) from T. thermophilus. D, Superposition of TtHICDH and closed-form IPMDH dimer (TfIPMDH, green) from T. ferrooxidans. Putative active-site pockets are marked with "active site" only for subunit A, except in panel B.

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FIG.2. Substrate-binding site of TtHICDH. A, Stereoview of the substrate-binding site of TtHICDH with electron density map. B, Superposition of the substrate-binding site of TtHICDH (hot pink) with isocitrate-bound ICDH from E. coli (cyan; PDB ID, 1AI2). Amino acid residues of TtHICDH and ICDH are indicated by bold and light letters, respectively. C, Superposition of the substrate-binding site of TtHICDH with 3-isopropylmalate-bound IPMDH from T. ferrooxidans (green; PDB ID, 1A05). Amino acid residues of TtHICDH and IPMDH are indicated by bold and light letters, respectively. Strand 3 and helix 4 are marked in panels B and C.

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FIG.3. Subunit interaction of TtHICDH. A, Overall structure of the TtHICDH tetramer. B, Stereoview of the electron density map in the interface. C, Close-up view of dimer-dimer interface. In panel C, a half of the amino acid residues involved in hydrophobic dimer-dimer interaction are shown. Subunits A, B, C, and D are colored by green, gold, hot pink, and blue, respectively.

Protein data bank accession number. The atomic coordinates and structure factors of TtHICDH have been deposited in the RCSB Protein Data Bank with accession number 1X0L.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Crystal structure of TtHICDH. TtHICDH crystallizes using PEG400 as the precipitant in the space group C222₁. There are two monomers in the crystallographic asymmetric unit (Table 1; Fig. 1A). The crystal diffracts to 1.85 Å. The three-dimensional structure of IPMDH from T. thermophilus HB8 with the Ala172Val mutation (25), which has 44% identity in amino acid sequence to TtHICDH, was used as the search model in molecular replacement. The final structure contains all the side chains in subunit A, but the side chains of the regions with residues 72 to 76 and 264 to 265 in subunit B are omitted because of the low electron density due to disorder in these regions. In addition, we could not find the substrate and Mg²⁺ ion that were added to the crystallization solution and cryoprotectant in the electron density map. Three hundred seventy-eight water molecules are contained in the asymmetric unit of the cell of TtHICDH. The final R factor of refined structure of TtHICDH is 21.6%, and free R is 24.8%.

The structure of the TtHICDH monomer is composed of two domains separated by the substrate-binding pocket and is very similar to those of IPMDH from T. thermophilus HB8 (TtIPMDH; PDB ID, 1IPD) (8), IPMDH from Thiobacillus ferrooxidans (TfIPMDH; PDB ID, 1A05) (7), and ICDH from Escherichia coli (EcICDH; PDB ID, 1AI2) (6), with root mean square deviations (RMSD) of 1.69 Å, 1.39 Å, and 1.65 Å for the -carbons, respectively (Fig. 1B). Although the crystal structure of TtHICDH contains neither isocitrate nor homoisocitrate in the putative substrate-binding site, the conformation of TtHICDH is more similar to that of substrate-bound TfIPMDH in a closed conformation (RMSD of 1.40 Å) than to that of TtIPMDH in an open conformation (RMSD of 1.72 Å) (Fig. 1B and C). This indicates that TtHICDH adopts a closed-like conformation even without bound substrate.

Substrate-binding site. Because of the better quality, we describe the structural detail only for the A subunit of TtHICDH. The closed-like conformation of the crystal structure of TtHICDH allowed us to elucidate the structural basis for the substrate specificity in TtHICDH by comparison of its structure with those of TfIPMDH and EcICDH, whose structures were determined in the closed forms. Homoisocitrate, isocitrate, and 3-isopropylmalate, which are the substrates of HICDH, ICDH, and IPMDH, respectively, contain a common malate moiety in their structures. The amino acid residues that recognize the moiety are conserved Arg88, Arg98, Arg118, Lys171, Asp204, Asp228, and Asp232 in TtHICDH and occupy spatial positions similar to those in EcICDH and TfIPMDH (Fig. 2B and C) (6, 7). Substrate-bound complex structures of ICDH and IPMDH revealed that the substrate specificity is determined by the residues in the loop between strand 3 and helix 4 (2, 5, 32). We previously reported that Arg85 is one of the most important residues for substrate recognition in TtHICDH (19). The crystal structure reveals that Arg85 is located in the substrate-binding pocket but extends its side chain toward Glu122 in another domain of the same subunit (Fig. 2A to C). The roles of Arg85 and Glu122 are described in detail in Discussion.

Subunit interface of TtHICDH. TtHICDH is a homotetrameric ß-decarboxylating dehydrogenase which is different from the generally homodimeric ß-decarboxylating dehydrogenases. In an asymmetric unit of the TtHICDH crystal, there are only two subunits. Considering the symmetry of the crystals, we could easily identify another intersubunit interaction (Fig. 3A). In the tetramer, two dimers have head-to-head interaction, with their arm regions composed of four ß-strands from a dimer. Interestingly, only a small number of hydrogen-bonding interactions contributes to the tetramer formation. On the other hand, the tetramer formation is highly stabilized by hydrophobic interaction. the Val135 mutant interacts with Tyr125 and Val141 mutants from a different subunit of the functional dimer and Tyr132 and Leu133 from another dimer (Fig. 3B and C).

Effects of mutation affecting the interdimeric interaction on the quaternary structure of TtHICDH. We isolated a mutant containing Val135Met during the course of a different experiment of directed evolution (we will report the results elsewhere in the near future). Since the mutant contained multiple amino acid substitutions, we constructed a mutant containing only a single Val135Met mutation. The quaternary structures of wild-type TtHICDH and the mutant were analyzed by two different methods, gel filtration and sedimentation equilibrium centrifugation. We first carried out molecular size calibration using a Hi-load Superdex200 prepgrade gel filtration column (Fig. 4). Elution volumes of TtHICDH and the Val135Met enzyme were 172.3 ml and 194.6 ml, respectively (Fig. 4A), corresponding to 3.21 and 1.74 subunits, respectively, in a single molecule (Fig. 4B). To confirm the quaternary structure further, we also analyzed the structure by sedimentation equilibration. Data were fitted best with the dimer-tetramer equilibrium for both wild-type TtHICDH and the Val135Met mutant (Table 2). However, TtHICDH is present mostly as a tetramer, and the Val135Met mutant is a dimer at 1.0 mg/ml. These results indicate that Val135 in TtHICDH plays a role in tetramerization in TtHICDH.

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FIG. 4. Gel filtration analysis of TtHICDHs. A, Elution profiles of TtHICDH, the Tyr125Ala mutant, and the Val135Met mutant in a Superdex200 prepgrade column; B, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of TtHICDH and the mutants. Lane 1, molecular mass markers; lane 2, purified TtHICDH; lane 3, purified Tyr125Ala protein; lane 3, purified Val135Met protein. Molecular mass markers used were phospholyrase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (20 kDa). C, Molecular size calibration using Superdex200 prepgrade gel filtration with a marker protein; 1, aldolase (158 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (43 kDa); 4, RNase A (13.7 kDa).

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TABLE 2. Quaternary structure of TtHICDH and mutants

Heat tolerance of TtHICDH and the mutants. Since the determined three-dimensional structure of TtHICDH suggests that tetramerization may contribute to its substantially higher thermal inactivation temperature, the thermotolerance of the mutant was analyzed by measuring the remaining activity after the purified enzymes were incubated at various temperatures for 20 min. Distinct activity (10%) remained even after incubation at 100°C for wild-type TtHICDH. On the other hand, the Val135Met mutant completely lost the activity after the incubation at 90°C (Fig. 5). Thermal-inactivation temperatures to show half of the initial activities were estimated to be approximately 97.6°C and 84.0°C for wild-type TtHICDH and the Val135Met mutant, respectively. These results indicate that tetramer formation plays a critical role in the extreme thermal-inactivation temperature.

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FIG. 5. Thermal inactivation of TtHICDHs. Remaining activity after heat treatment at appropriate temperatures for 20 min. In each figure, TtHICDH, the Tyr125Ala mutant, and the Val135Met mutant are indicated by open circles, open diamonds, and open squares, respectively.

Kinetic properties of TtHICDH and the mutants. Because of the shortage of homoisocitrate, which is not commercially available now, in our reagent stock, we carried out the kinetic assay of the enzymes using isocitrate as a substrate. The K_m values of TtHICDH and the Val135Met mutant for isocitrate were 521 ± 42 µM and 161 ± 18 µM, respectively, and the k_cat values were 438 ± 14 s^–1 and 211 ± 6 s^–1 for TtHICDH and the Val135Met mutant, respectively (Table 3). When calculated per monomer, the k_cat values for the wild type and mutant were 110 and 106, respectively, indicating that the catalytic mechanism in TtHICDH is the same in the Val135Met mutant. The k_cat/K_m values, showing the catalytic efficiency, for isocitrate are 0.84 s^–1 M^–1 and 1.3 s^–1 M^–1, indicating that the activity is enhanced by 1.6-fold through tetramer-to-dimer structural transition generated by the Val135Met mutant and that the improvement is attributed to the decrease in K_m for the substrate.

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TABLE 3. Kinetic parameters of TtHICDH and mutants

Effect of mutation at Tyr125 on catalytic activity and tetramer formation. The Tyr residues Tyr140 and Tyr160 in TfIPMDH and EcICDH, respectively, are conserved among related ß-decarboxylating dehydrogenases. In the crystal structures of TfIPMDH and EcICDH, the Tyr residue extends its side chain toward the substrate to perform dehydrogenation of the substrate and/or protonation of the substrate after decarboxylation (11). A Tyr residue (Tyr125) is conserved at the corresponding position in the primary sequence of TtHICDH. However, the Tyr residue extends its side chain in the opposite direction (Fig. 2B and C), contributing to the hydrophobic interaction necessary for tetramer formation (Fig. 3). To elucidate the role of Tyr125 in catalytic function, tetramer formation, and thermostability in TtHICDH, we constructed the mutant TtHICDH with replacement of Tyr125 by Ala. Kinetic analysis showed that the mutation significantly decreased the k_cat value (0.1 s^–1) by eightfold but that the K_m value for isocitrate slightly increased (707 ± 97 µM) (Table 3). The result suggests that Tyr125 is involved in the catalytic process of this enzyme. On the other hand, analysis of the quaternary structure of the mutant by both gel filtration and sedimentation equilibrium analysis (Fig. 4; Table 2) showed that the mutant is a homotetramer since the elution volume was 164.5 ml, corresponding to 4.00 subunits organized in a single molecule and since only a monomer-tetramer model fit with the sedimentation equilibrium. The mutant showed a thermal inactivation curve similar to that of wild-type TtHICDH (Fig. 5). These results indicate that the mutation affected neither the oligomeric state nor the thermotolerance of TtHICDH but affected catalytic function.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In our previous study, we showed that the substrate specificity of TtHICDH was dramatically altered by the Arg85Val mutation (19). The Arg85Val mutant lost activity towards isocitrate but interestingly showed activity towards 3-isopropylmalate, the substrate of IPMDH that is not normally recognized by TtHICDH. Superposition of the structure of TtHICDH with those of EcICDH and TfIPMDH indicates that Arg85 is found at a position close to the -moiety of the substrates, occupies a position similar to that of the corresponding Leu92 in TfIPMDH, and is positioned 1.6 Å (for -carbons) away from Val116, the counterpart of Arg85, in EcICDH (Fig. 2B and C). Therefore, we assume that replacement of Arg85 with Val may increase hydrophobicity in the region, which may facilitate binding of 3-isopropylmalate and prevent isocitrate from binding.

The crystal structure of TtHICDH indicates that the guanidyl group of the Arg85 side chain interacts with Glu122 from another domain in the same subunit. Glu122 is highly conserved in the primary structures of most HICDHs, ICDHs, and archaeal IPMDHs, although its function has not yet been characterized. In EcICDH, the equivalent residue, Glu157, interacts with the main-chain nitrogen of Phe190 in the same domain and is not directed towards the substrate-binding site in both apo and holo forms of EcICDH and TtIPMDH (8, 11) (Fig. 2B and C). Therefore, it is unlikely that Glu157 in EcICDH is involved in catalysis. In TtHICDH, however, Glu122 is flipped towards the substrate-binding site to interact with Arg85 electrostatically (Fig. 2A to C).

Another feature characteristic of the TtHICDH structure is found at Tyr125, a conserved residue among related ß-decarboxylating dehydrogenases. The phenolic side chain of Tyr125 occupies a position totally different from those of the corresponding Tyr residues in TfIPMDH and EcICDH. Nevertheless, the Tyr125Ala mutation caused a substantial decrease in the k_cat value, as do corresponding mutations in EcICDH (Tyr160) and TtIPMDH (Tyr140) (11, 15, 21). This result suggests that Tyr125 is involved in the catalytic function of TtHICDH. If the unusual orientation of Glu122 and Tyr125 is present under physiological conditions, we speculate that substrate binding causes a conformational change of the region containing Glu122 and Tyr125 in TtHICDH as follows, based on the crystal structure of TtHICDH and information on the mutation effect of Tyr125. In this scenario, when a substrate with a negatively charged -moiety of a substrate, such as homoisocitrate and isocitrate, binds to the substrate-binding site, the interaction between Arg85 and Glu122 in the apo form may be lost due to repulsion between the negative charges of the substrate -moiety and Glu122. The positive charge of Arg85 may interact with a carboxylate of the substrate -moiety. Glu122 then rotates its side chain to form a hydrogen bond, possibly with a main-chain nitrogen in the case of EcICDH. Accompanied by the Glu122 rotation, Tyr125 located on the same loop may be delivered to the substrate-binding site to play the catalytic role. Since the Tyr residue takes the same position in ICDH and IPMDH even in an apo form (7, 15), it is unlikely that the Tyr residue changes the orientation upon substrate binding in other ß-decarboxylating dehydrogenases. It should be noted that the pH level used for the crystallization of TtHICDH was 4.8, which was lower than those for other ß-decarboxylating dehydrogenases (pHs 5.4 to 7.5). This may suggest the possibility that the unusual orientation of Glu122 and Tyr125 may not be seen under physiological conditions. In any case, to understand the substrate recognition mechanism, X-ray analysis of substrate-bound complex is obviously required.

TtHICDH is a tetrameric enzyme with extremely high thermotolerance in comparison with TtICDH and TtIPMDH (12, 19). The Val135Met mutation causes a significant decrease in the thermotolerance, accompanied by the tetramer-to-dimer transition of the quaternary structure (Fig. 5). Therefore, these results indicate that the tetramer formation is one of the major factors ensuring the extremely high thermotolerance of this enzyme. Val135 contributes to a hydrophobic environment with Tyr125 and Val141 from another subunit of the same dimer and Tyr132 and Leu133 from another dimer (Fig. 3B and C). The Tyr125Ala mutation would also weaken the hydrophobic interaction in the dimer-dimer interface. However, the mutation apparently does not preclude tetramerization of the enzyme (Fig. 4A and C; Table 2). Although the Tyr125Ala mutation was introduced at the position close to Val135, the mutant still possesses high thermotolerance (Fig. 5). We assume that the large side chain of Met introduced at position 135 would hinder the correct hydrophobic interaction between the residues and therefore prevented TtHICDH from forming a tetramer, leading to lower thermotolerance. It should be noted that the activity of the dimeric TtHICDH mutant (the Val135Met mutant) is 1.6-fold higher than that of the tetrameric wild-type TtHICDH because of the improved K_m value for isocitrate (Table 3), indicating that tetramerization of TtHICDH may sacrifice some activity for obtaining higher thermotolerance.

It is known that ICDH from Thermotoga maritima (26) and IPMDH from Sulfolobus tokodaii 7 (StIPMDH) (27, 33) are also tetrameric ß-decarboxylating dehydrogenases, although most ß-decarboxylating dehydrogenases are dimers. Interestingly, the tetrameric ß-decarboxylating dehydrogenases are found only in hyperthermophiles and not in mesophiles. ICDHs from two hyperthermophilic archaea, Pyrococcus furiosus and Aeropyrum pernix, are dimeric enzymes having thermostability (melting temperatures, >100°C) higher than that of the tetrameric ICDH from Thermotoga maritima, StIPMDH, and TtHICDH (26). In TtHICDH, the arm region forming the ß-sheet is responsible for the tetramerization. However, in ICDH from Aeropyrum pernix and EcICDH (6, 10), the arm region contains an additional -helix and interacts with the same region from another subunit to form a small domain named clasp, which may prevent these ICDHs from forming a tetramer. Thus, the fact that two hyperthermophilic ICDHs are dimeric indicates that tetramerization is not the only way to acquire elevated thermostability. In tetrameric StIPMDH, the hydrophobic residues that correspond to Val135, Tyr132, and Val141 in TtHICDH are conserved as Val135, Val131, and Ile141, respectively, indicating that this enzyme may have the similar hydrophobic dimer-dimer interactions to form a tetramer (27). The crystal structure of StIPMDH at a 2.8-Å resolution (which has recently been deposited in the RCSB Protein Data Bank as 1WPW) indicates that StIPMDH is also stabilized by a dimer-dimer interaction like that of TtHICDH. On the other hand, although ICDH from T. maritima is also a tetrameric ß-decarboxylating dehydrogenase (26), its amino acid sequence in the arm region does not align well with those of TtHICDH and StIPMDH, suggesting that the enzyme has a different strategy to form a tetramer. Considering that TtHICDH possesses ancestral features of ß-decarboxylating dehydrogenases, through billions of years of evolution, the role of the arm region for tetramerization to enhance thermostability may have been lost to enhance the activity for ß-decarboxylating dehydrogenation, especially in mesophilic organisms (Table 3).

In summary, TtHICDH, while structurally similar to other members of its family, forms a tetramer that is more thermotolerant than the Val135Met mutant, which forms dimers. Tyr125 forming the active site is found in a conformation that is likely to change upon substrate binding and/or under physiological conditions. The observation that the Val135Met mutant is catalytically more active at the expense of thermotolerance suggests that other related enzymes may have evolved that way.

 
We thank the staff of the Photon Factory for their assistance with the data collection. The data collection was approved by the Photon Factory Advisory Committee (proposal no. 2003G103). We also thank Elinor T. Adman (Washington University School of Medicine) for providing us useful comments for completing the manuscript.

This work was supported in part by grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from Noda Institute for Scientific Research, and from the Nagase Science and Technology Foundation.

 
* Corresponding author. Mailing address: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81-3-5841-3074. Fax: 81-3-5841-8030. E-mail: umanis{at}mail.ecc.u-tokyo.ac.jp.

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Journal of Bacteriology, October 2005, p. 6779-6788, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6779-6788.2005
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